Access to cellular components such as nucleic acids is imperative to a variety of molecular biology methodologies. Such methodologies include nucleic acid sequencing, direct detection of particular nucleic acid sequences by nucleic acid hybridization and nucleic acid sequence amplification techniques.
Although access to nucleic acids from the cells of some organisms does not involve particularly complex methodologies or harsh treatments, other organisms have cells from which it is particularly difficult to access nucleic acids or other cellular components. Organisms in the latter group include species of the genus Mycobacteria, yeast and fungi. Usually, the difficulty in cellular component access is a result of organism cell walls which are highly resistant to lysis or disruption, and/or the adherence of certain cellular components such as nucleic acids to cellular proteins and other cellular substances such as pieces of cell walls.
Due to the difficulties in attempting to access nucleic acids from mycobacterial organisms the methods utilized tend to be harsh and thus not very useful with non-mycobacterial organisms. Conversely, the methods used to disrupt cells and access nucleic acids from non-mycobacterial organisms are often not effective when used with mycobacterial organisms.
Two non-enzymatic methods which have been used to disrupt cells to access nucleic acids are the application of heat to cells (see U.S. Pat. No. 5,376,527) and physical agitation of cells in the presence of lysogenic chemicals with or without "minibeads". For example, DeWitt et al., J. Clin. Micro. 28 (11):2437-2441 (1990) describe the orbital shaking of samples containing mycobacterial cells in the presence of buffered phenol and sodium dodecyl sulfate (SDS), Hurley, S. S. et al., J. Clin. Microbiol 25 (11) 2227-2229 (1987) describe a combination of phenol extraction and physical rupture of mycobacterial cells with zirconium beads in a Biospec Products Mini-Beadbeater, and Shah, J. S. et al., J. Clin Microbiol 33 (2), 322-328 (1995) describe the lysis of heat-inactivated mycobacterial cells with the lysogenic agent guanidimium thiocyanate (GuSCN) and physical agitation with zirconium oxide beads. Also, U.S. Pat. No. 5,374,522 describes methods of disrupting cells by applying ultrasonic energy to the cells in the presence of beads, and Hurley et al., Int. J. Systemic Bacteriology 38 (2):143-146 (1988) describe physical agitation of samples containing mycobacterial cells in the presence of distilled phenol and zirconium beads.
As recognized in U.S. Pat. No. 5,374,522, rigorous physical grinding or shaking of organisms whether with or without beads presents considerable drawbacks. First, friction resulting from the physical interaction of grinding particles can create excessive heat which has deleterious effects on nucleic acids, and thus can render the nucleic acids unusable in subsequent hybridization procedures. Also, many of the organisms whose cells require such harsh conditions for extraction of cellular components are extremely pathogenic, and thus present health hazards when subjected to these physical manipulations in an open system. Also, the use of lysogenic chemical agents and/or enzymes such as SDS, GuSCN, proteinases, phenol/chloroform, etc. often adversely affects subsequent molecular biology processes for which the nucleic acids are accessed. For example, ionic and non-ionic detergents are known to inhibit nucleic acid amplification processes such as polymerase chain reaction (PCR) and strand displacement amplification (SDA), and carbon black which is commonly used to process glass beads is known to inhibit SDA.